Download Research Paper Synthesis and Evaluation of Clozapine

International Journal of Pharmaceutical Sciences and Nanotechnology
Volume 2 • Issue 4 • January – March 2010
Research Paper
Synthesis and Evaluation of Clozapine and its Related Compounds
Nagaraju Garipelli1,*, B Madhava Reddy1, Jithan AV2
1
G Pulla Reddy College of Pharmacy, Hyderabad, AP, India
Vaagdevi College of Pharmacy, Hanamkonda, Warangal, AP, India
2
ABSTRACT:
Clozapine (8-chloro-11- (4-methyl-1-piperazinyl)-5H-dibenzo[b,e][1,4]diazepine) is a dibenzodiazepine
derivative, used in the treatment of schizophrenia. The present work aimed to develop a new modified method for the
synthesis of clozapine and also impurity profiling which is mandatory by the regulatory bodies. Present synthetic method for
clozapine has the advantages over the previous processes in respect of safety, cost and efficiency. Impurity profiling
indicated that 8-chloro-10, 11-dihydro-11-oxo-5H-dibenzo [b, e] [1, 4] diazepine, clozapine dimer, desmethyl clozapine, and
clozapine N-oxide are very minor impurities in the synthesis of Clozapine. These compounds were confirmed by analytical
data. The impurities were also synthesized and evaluated for the neuropharmacological activity. The study revealed that
three of four impurities have decreased the locomotor activity in mice, which indicates the similar activity compared to
clozapine as antidepressant
KEYWORDS:
Clozapine, impurities, synthesis, locomotor activity.
Introduction
Clozapine (8-chloro-11-(4-methyl-1-piperazinyl)-5Hdibenzo [b,e][1,4]diazepine) is classified as an 'atypical'
antipsychotic drug (Ioulia Savina et al., 2007). It is a
yellow powder with a molecular weight of 326.83 and
melting point of 182ºC. It is synthesized using very routine
synthetic procedures. However, the current existing
procedures of the synthesis involve the use of a few toxic
chemicals and tedious workup processes. In this study, we
aimed to develop a newer synthesis for Clozapine. The
second objective of this project was to conduct impurity
profiling on the newly developed synthetic process.
Recently the pharmacopoeiae are specifying the nature of
impurities in the monograph of drug products and the
dosage manufacturers are also intended to have the
impurity profile of the drug substance (Sharp TR et al.,
2001, ICH., 2000). Therefore it was proposed to study the
impurity profile of Clozapine (identifying the impurities by
HPLC methods), synthesis and characterization of these
impurities by spectral data and screening of the impurities
(Alsante KM et al., 2001) for neuropharmacological
activity along with the Clozapine.
Schizophrenia is a severe, idiopathic psychiatric disorder
with a polygenic component. It is composed of severe
thought disorders, termed psychoses, which are
characterized by illogical, delusional, or paranoid thoughts
(Jaine N Delgado et al., 1998). Schizophrenia is the fourth
largest disease in the world in the age group of 15-30 and
posing it as one of the major medical challenges. The
medical management of schizophrenia often requires a
combination of antipsychotic, antidepressant, and
antianxiety drugs (Helio Elkis., 2007). Disruption of the
dopamine system has been strongly linked to
Schizophrenia and related psychosis, with abnormally high
dopamine action apparently leading to these conditions.
Numbers of drugs having the activity for blocking the
neurotransmission of dopaminergic receptor in the central
nervous system (Ross JB et al., 1996) have been
developed. These include phenothiazine type compounds
such as chlorpromazine and butyrophenone derivatives,
and benzamide type compounds such as sulpiride. The
newer antipsychotic drugs, without substantial risk of extra
pyramidal effects (John L Neumeyer et al., 2000) are
referred to as atypical neuroleptics. The term atypical
refers to the mechanism of antipsychotic action other than
or in addition to postsynaptic D2 receptor blockade.
Atypical agents are clozapine, Resperidone, Aripiprazole,
Quetiapine, and Olanzapine (Umit B et. al, 2007).
Materials and Methods
All the chemicals used were of synthetic grade and
acquired from Sigma-Aldrich distributor. Melting points
were recorded on Polmon melting point apparatus in open
capillary tubes and were uncorrected. Reactions were
monitored with the aid of TLC precoated silica gel G
plates. The IR spectra were recorded on Perkin Elmer 882
* For correspondence: Nagaraju Garipelli
E-mail: [email protected]
762
Nagaraju Garipelli, et al. : Synthesis and Evaluation of Clozapine and its Related Compounds
FT-IR spectrophotometer by using 1% KBr technique and
H1-NMR spectra are obtained
in CDCl3 on Brukar
Avance 300 MHz relative to TMS as internal standard
respectively. The Electron Ionization mass spectrum was
recorded on Agilent 1100 series LC-MSD-TRAP-SL
system. Purity of the compounds was determined by the
Waters HPLC system using C-18 column. Pharmacological
evaluation was carried out on adult male albino mice (20-
30 g). These were procured from Mahaveer Enterprises,
Hyderabad, India. The animals were fed with standard
pellet diet and water ad libitum. All the animals were
acclimatized a week before use. The locomotor activity
was determined by Digital Photoactometer (M.K.M.,
Chennai). Chemicals used were Clozapine (Clozaril®,
Novartis), Clozapine related compounds, and dimethyl
sulphoxide (DMSO) are of standard grades.
Scheme-I
Proposed route of synthesis
Cl
H
N
O
Cl
N
H
N
Cl
TiCl4
763
110-112 0C
Toluene, anisole
N
H
Reflux for 3hrs
8-chloro-11oxo-10,
11-dihydro-5H-dibenzo-1,
4-diazepine
CH 3
N
10 0 C
N
H
1-Methylpiperazine e
1-Methyl-piperazin
Reflux for 4 hrs
CLOZAPIN E (crude)
Recrystallization
from
acetone-IPE
CH 3
N
N
Cl
N
N
H
CLOZAPIN E
764
Volume 2 • Issue 4 • January -March 2010
International Journal of Pharmaceutical Sciences and Nanotechnology
Scheme-II
Synthesis of Clozapine Dimer and Desmethyl Clozapine
O
H
N
Cl
H
N
Ticl4/toluene
1,4 -dio xane
+
110-1120 C
N
H
N
H
Reflux for 4hrs
Reflux for 3hrs
Piperazine
8-chloro-11oxo-10,11dihydro
-5H-dibenzo-1,4diazepine
NH
H
N
N
N
Cl
Cl
N
+
N
N
H
N
N
Cl
Desmethyl clozapine
N
H
Clozapine dimer
Scheme-III
Synthesis of Clozapine N-Oxide
CH3
CH 3
N
N
N
Cl
N
N
N
H
O
Cl
mCPBA/MDC
Clozapine
Synthesis of Clozapine and its related compounds
Clozapine
Scheme–I was used for the synthesis of Clozapine. In the
Scheme-I, Toluene, 200ml, anisole 20ml and 8-Chloro-11-
N
N
H
Clozapine N-Oxide
oxo-10, 11-dihydro-5H-dibenzo-1, 4-diazepine (10 g)
charged in round bottom flask at RT and stirred for 30 min.
The reaction mass cooled to 10-15oC and 9.7 ml (16.4 g)
TiCl4 was added drop wise for 30 mins with constant
stirring. The reaction mixture was heated to 110-112oC and
heated under reflux for 3 hr. The reaction mass was then
Nagaraju Garipelli, et al. : Synthesis and Evaluation of Clozapine and its Related Compounds
cooled to 10oC. Methyl piperazine 20 g was added to the
reaction mass during next 30 min, and further it was
continuously stirred for 30 mins at 100C. The reaction
mixture was then heated under reflux for 4 hr. Progress of
reaction was checked by thin layer chromatography (TLC)
using mobile phase chloroform and methanol (9:1). After
confirmation the reaction mass was distilled off to get the
residue. The residue was partitioned between ethyl acetate
and 2M aqueous hydrochloric acid (300 ml each) and
filtered under vacuum. Both layers were separated and the
aqueous layer was washed with ethyl acetate (2X150ml)
and basified with sodium hydroxide solution to pH 14. The
basified aq. layer extracted with ethyl acetate (3X150ml).
Ethyl acetate layer was washed with DM water (2X100ml)
and then with brine solution (100ml) and dried over
enhydrous sodium sulphate. The ethyl acetate layer was
completely distilled off under vacuum below 40oC to get
clozapine. This method has advantages over the previous
methods in terms of time, solvent consumption and safety.
765
piperazine (25 g) was added to the reaction mass over 30
min. for desmethyl clozapine, for clozapine dimer, (15 g)
and continued the stirring for 30 min. at 100C. The reaction
mixture heated under reflux for 4 hr. Progress of the
reaction checked by thin layer chromatography (TLC)
using mobile phase chloroform and methanol (9:1). After
confirmation by TLC, reaction mass completely distilled
off to get residue. The residue partitioned between ethyl
acetate and 2M aqueous hydrochloric acid (300 ml each)
and filtered under vacuum. Both layers separated and the
aqueous layer washed with ethyl acetate (2X150ml) and
basified with sodium hydroxide (NaOH) to pH 14. The
basified aq. layer extracted with ethyl acetate (3X150ml).
Ethyl acetate layer washed with DM water (2X100ml) and
then with brine solution (100ml) and dried over enhydrous
sodium sulphate. The ethyl acetate layer completely
distilled off under vacuum below 40oC to get the
clozapine.
Synthesis of Clozapine N-oxide
Impurities
1) 8-Chloro-11oxo-10, 11-dihydro-5H-dibenzo-1, 4diazepine, 2) Clozapine Dimer, 3) N-desmethyl clozapine
or Norclozapine, 4) Clozapine N-oxide (Capuano et al.,
1999, 2002) are the impurities in this synthesis of Clozapine.
Preparation of Clozapine Dimer and Desmethyl
clozapine
In Scheme-II, of Toluene 200ml, anisole 20ml and 8Chloro-11-oxo-10, 11-dihydro-5H-dibenzo-1, 4-diazepine
(10 g) charged in round bottom flask at RT and stirred for
30 min. The reaction mass cooled to 10-15oC and TiCl4
9.7 ml (16.4 g) added drop-wise over 30 min. with stirring.
The reaction mixture was heated to 110-112oC and
refluxed for 3 hr. The reaction mass cooled to 10oC.
In Schme-III, Clozapine (10 g) and MDC (200ml) were
charged into round bottom flask at RT, with stirring. The
reaction mixture was cooled to 0oC and meta-chloro
perbenzoic acid (mCPBA 15g) in methylene dichloride
(MDC) drop-wise over 1 hr with stirring. Progress of the
reaction checked by thin layer chromatography (TLC) with
mobile phase consisting of chloroform and methanol (9:1
ratio). Temperature was raised to 150C and pH adjusted to
7 by saturated sodium bicarbonate solution and both layers
were separated. MDC layer was washed with DM water
and (2X200ml) dried over enhydrous sodium sulphate. The
MDC layer completely distilled off under vacuum below
40oC.
Table 2. Spectral data Analysis for Clozapine and its impurities
Compound
IR (cm-1)
NMR (in CDCl3, δ)
Mass (m/z)
Clozapine
2968(aliphatic C-H str of CH3),
3290(NH str), 1594(C=N str), 1255
(C-N ben), 778 (C-Cl str)
7.2-6.5 (m, aromatic 7H), 4.8 (s, H of
NH,), 3.4-2.5 (t, 8H of CH2), 2.3 (s, 3H
of CH3)
327(M+),
329(M+2)
Imp-A
3059 (aliphatic C-H str of CH3), 3181
(NH str), 1220 (C-N ben),
749 (C-Cl str)
7.2-6.5 (m, aromatic 7H), 4.8 (s, H of
NH,)
245 (M+),
511 (dimer),
753 (trimer)
Desmethyl clozapine
3257 (NH str), 1602 (C=N str),
1296 (C-N ben), 759 (C-Cl str)
7.3-6.5 (m, aromatic 7H), 4.89 (s, 2H of
NH,), 3.5-3 (t, 8H of CH2)
313(M+),
315(M+2)
Clozapine dimer
2920 (aliphatic C-H str of CH3), 3277
(NH str), 1599 (C=N str),
1251 (C-N ben), 757 (C-Cl str)
7.3-6.6 (m, aromatic 7H), 4.89 (s, H of
NH,), 3.5 (t, 8H of CH2)
539(M+),
541(M+2),
542(M+3)
766
International Journal of Pharmaceutical Sciences and Nanotechnology
Pharmacological Evaluation
Evaluation of Neuropharmacological activity
Clozapine is an antipsychotic drug act by decreasing the
levels of dopamine, by antagonizing the dopamine
receptors in central nervous system. It binds to not only
dopamine receptors but also to various receptors in brain
and elicits different neurological effects. Clozapine and the
impurities are screened for the locomotor activity (Jelveh
Lameh et al., 2007).
Locomotor Activity
Adult male albino mice (20-30 g) were divided into 3
groups. Group I (control) animals were administered only
vehicle (DMSO), 0.5ml, i.p. Group II (standard) animals
were administrated (i. p.) drugs, Clozapine in a dose of 2.5
mg/kg of body weight (Prus AJ et al., 2005). Group III-IX
Volume 2 • Issue 4 • January -March 2010
animals were administered by the impurities, through i.p.
in 2.5 and 5 mg/kg body weight. All the groups of animals
were tested after 10 min. for activity in photoactometer for
10 min. The basal activity scores of all animals were taken.
Clozapine (0.5mg/kg, i.p.) injected and after 10 min retested each mouse for activity scores for 10 min.
Difference in the activity, before and after drug
administration calculated in terms of percentage decrease
in motor activity (Khan et al., 2004).
All the impurities have decreased the scores in the
actophotometer compared to the 0th minute (before
administration of the impurities) indicating that they
exhisit the locomotor activity similar to that of clozapine at
both the tested doses (2.5 mg& 5 mg/kg); desmethyl
clozapine being more closer to clozapine. The scores and
the percentage inhibition of the locomotor activity are
presented a in Table 3 and 4 respectively.
Table 3. Locomotor activity of Clozapine and its impurities
(scores)
Time
Control
Standard
(min)
(DMSO)
(Clozapine)
Imp-A
2.5 mg/kg
Desmethyl clozapine
2.5 mg/kg
5 mg/kg
2.5 mg/kg
5 mg/kg
Clozapine
Clozapine dimer
N-Oxide
(5mg/kg) 2.5 mg/kg 5 mg/kg
0
246.7±10.04 230.4±12.89
227.5±4.1
224±5.5
239±7.91
246.1±6.4
275±4.2
239.6±3.2 240.8±4.3
10
231.7±7.34
158.7±7.4
114.6±6.1
128.6±3.5
124.5±6.02
252±11.7
125.8±4.5 122.3±4.1
118.6±11.38
Values are mean ± SD (n=6);
Table 4. Percentage inhibition of locomotor activity by clozapine and its impurities
Group
Treatment
Dose (mg/kg)
Inhibition (%)
I
Control
-
6.05±3.13
II
Clozapine
2.5
48.5±3.6a
III
Imp-A
2.5
30.2±3.13 a
IV
Imp-A
5
48.8±2.57a
V
Desmethyl Clozapine
2.5
46.1±6.3 a
VI
Desmethyl Clozapine
5
49.4±6.6 a
VII
Clozapine-N-oxide
5
8.3±4.14
VIII
Clozapine dimer
2.5
47.4±5.12 a
IX
Clozapine dimer
5
49.2±1.75 a
Values are mean ± SD (n=6); a p<0.001 Vs Control
Nagaraju Garipelli, et al. : Synthesis and Evaluation of Clozapine and its Related Compounds
risperidone or typical antipsychotic medications. Schiz
Res. 94(1-3): 128-138(2007).
Discussion
Clozapine is a dibenzodiazepine derivative having
neurotransmission blocking activity for dopaminergic
receptors and used in the treatment of schizophrenia. The
prior methods of synthesis of Clozapine involve the use of
few toxic chemicals and tedious workup procedures. In
one of the reported synthetic process (US Patent –
3962248), ether is used for extraction and ammonia for
basification. In another process (US Patent – 3539573)
phosphoroxycloride and benzene are used. This present
synthetic process, it avoid the use of above toxic chemicals
and makes use of less toxic and safe chemicals like TiCl4
and ethyl acetate. The impurity profiling for this synthetic
process by HPLC has revealed four impurities viz; starting
material (8-chloro-11oxo-10, 11-dihydro-5H-dibenzo-1, 4diazepine). Clozapine dimmer, Desmethyl Clozapine and
Clozapine N oxide. All these impurities are structurally
very close to clozapine. Therefore, these impurities were
also synthesized and evaluated for locomotor activity along
with clozapine, with a view to find whether they also
possess similar activity. The locomotor activity screening
has shown that all the impurities possess similar kind of
activity. However, the magnitude of the activity is less
than clozapine except the desmethyl clozapine which is
almost equal to clozapine. Therefore, it can be concluded
that the presence of these impurities in the clozapine bulk
active chemical do not alter the pharmacological profile of
the clozapine drug to any greater extent.
Acknowledgments
The authors are thankful to G. Pulla Reddy College of
Pharmacy, Hyderabad, AICTE, New Delhi, and Matrix
Laboratories, Hyderabad.
References
Helio Elkis, Treatment Resistant Schizophrenia. Psychiatr
Clin North Am. 30(3): 511-533(2007).
Umit B. Semiz, Mesut Cetin, Cengiz Basoglu, Servet
Ebrinc, Ozcan Uzun, Hasan Herken, Hakan Balibey,
Ayhan Algul and Alpay Ates, Clinical predictors of
therapeutic response to clozapine in a sample of
Turkish
patients
with
treatment-resistant
schizophrenia.
Progress
in
NeuroPsychopharmacology
and
BiologicalPsychiatry. 31(6):1330-1336(2007).
Jelveh Lameh, Ethan S Burstein, Eve Taylor, David M
Weiner, Kimberly E Vanover and Douglas W
Bonhaus. Pharmacology of N-desmethylclozapine
Pharmacology
&
Therapeutics.
115(2): 223231(2007).
Ioulia Savina and Richard J Beninger. Schizophrenic
patients treated with clozapine or olanzapine perform
better on theory of mind tasks than those treated with
767
John L Neumeyer and Raymond G.Booth. Neuroleptics
and Anxiolytic agents. In WilliamO. Foye, Thomas
L.Lemke, David A. Willaims (4thedn), Principles of
Medicinal Chemistry, B.I.Waverly pvt. Ltd, New
Delhi, 2000, pp199-232.
Jaine N Delgado, Willaim A Remers. Antipsychotics, In
Wilson and Gisvold’s Text book of Organic Medicinal
and Pharmaceutical Chemistry (10thedn.), LipincottRaven, Philadelphia, 1998, pp. 449-454.
Ross JB. Drugs in the treatment of psychiatric disorders. In
Goodman and Gilman’s The pharmacological basis of
therapeutics (9thedn), McGraw-hill, New Jersey, 1996,
pp.399-424.
Khan and Khanum. Fundamentals of Biostatistics, Ukaaz
Publishers, 2004.
Capuano B, Crosby IT, Lloyd EJ, and Taylor DA.
Synthesis
and
Preliminary
Pharmacological
Evaluation of 4' Arylmethyl Analogues of Clozapine.
I. The Effect of Aromatic Substituents. Aust. J. Chem,
55: 565-576(2002).
Capuano B, Crosby IT, Lloyd EJ. Schizophrenia: Genesis,
Receptorology and Current Therapeutics. Curr. Med.
Chem. 9(5): 521-548(2002).
Prus
AJ, Philibin SD, Pehrson, AL, Porter JH.
Generalization to atypical antipsychotic drugs depends
on training dose in rats trained to discriminate 1.25
mg/kg clozapine versus 5.0 mg/kg clozapine versus
vehicle in a three-choice drug discrimination task.
Behavioural Pharmacology. 16(7): 511-520(2005).
Capuano B. 8-chloro-11oxo-10, 11-dihydro-5H-dibenzo-1,
4-diazepine. Molecules. 4: 329-332(1999).
Alsante KM, Hatajik TD, Lohr LL, and Sharp TR.
Isolation and identification of process related
impurities
and
degradation
products
from
pharmaceutical drug Candidates. Part 1. American
Pharmaceutical Review. 4(1): 70-78(2001).
Sharp TR, Alsante KM, and Hatajik TD. Isolation and
identification of process related impurities and
degradation Products from pharmaceutical drug
Candidates Part II: The roles of NMR and Mass
Spectrometry. American Pharmaceutical Review. Fall
issue 2001
International Conferences on Harmonization, Draft
Revised Guidance on Impurities in New Drug
Products. Q3B(R). Federal Register. 65(139): 4479144797(2000).
International Conferences on Harmonization, Draft
Revised Guidance on Impurities in New Drug
Substances. Q3A(R). Federal Register. 65(140):
45085-45090(2000).